β-Adrenergic receptor-induced activation of nerve growth factor gene transcription in rat cerebral cortex involves CCAAT/enhancer-binding protein δ

Stimulation of β-adrenergic receptors (BAR) by clenbuterol (CLE) increases nerve growth factor (NGF) biosynthesis in the rat cerebral cortex but not in other regions of the brain. We have explored the transcription mechanisms that may account for the cortex-specific activation of the NGF gene. Although the NGF promoter contains an AP-1 element, AP-1-binding activity in the cerebral cortex was not induced by CLE, suggesting that other transcription factors govern the brain area-specific induction of NGF. Because BAR activation increases cAMP levels, we examined the role of CCAAT/enhancer-binding proteins (C/EBP), some of which are known to be cAMP-inducible. In C6–2B glioma cells, whose NGF expression is induced by BAR agonists, (i) CLE increased C/EBPδ-binding activity, (ii) NGF mRNA levels were increased by overexpressing C/EBPδ, and (iii) C/EBPδ increased the activity of an NGF promoter–reporter construct. Moreover, DNase footprinting and deletion analyses identified a C/EBPδ site in the proximal region of the NGF promoter. C/EBPδ appears to be responsible for the BAR-mediated activation of the NGF gene in vivo, since CLE elicited a time-dependent increase in C/EBPδ-binding activity in the cerebral cortex only. Our data suggest that, while AP-1 may regulate basal levels of NGF expression, C/EBPδ is a critical component determining the area-specific expression of NGF in response to BAR stimulation.

A response regulator of cyanobacteria integrates diverse environmental signals and is critical for survival under extreme conditions

Microorganisms must sense their environment and rapidly tune their metabolism to ambient conditions to efficiently use available resources. We have identified a gene encoding a response regulator, NblR, that complements a cyanobacterial mutant unable to degrade its light-harvesting complex (phycobilisome), in response to nutrient deprivation. Cells of the nblR mutant (i) have more phycobilisomes than wild-type cells during nutrient-replete growth, (ii) do not degrade phycobilisomes during sulfur, nitrogen, or phosphorus limitation, (iii) cannot properly modulate the phycobilisome level during exposure to high light, and (iv) die rapidly when starved for either sulfur or nitrogen, or when exposed to high light. Apart from regulation of phycobilisome degradation, NblR modulates additional functions critical for cell survival during nutrient-limited and high-light conditions. NblR does not appear to be involved in acclimation responses that occur only during a specific nutrient limitation. In contrast, it controls at least some of the general acclimation responses; those that occur during any of a number of different stress conditions. NblR plays a pivotal role in integrating different environmental signals that link the metabolism of the cell to light harvesting capabilities and the activities of the photosynthetic apparatus; this modulation is critical for cell survival.

Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II

The classically recognized functions of the renin–angiotensin system are mediated by type 1 (AT1) angiotensin receptors. Whereas man possesses a single AT1 receptor, there are two AT1 receptor isoforms in rodents (AT1A and AT1B) that are products of separate genes (Agtr1a and Agtr1b). We have generated mice lacking AT1B (Agtr1b −/−) and both AT1A and AT1B receptors (Agtr1a −/−Agtr1b −/−). Agtr1b −/− mice are healthy, without an abnormal phenotype. In contrast, Agtr1a −/−Agtr1b −/− mice have diminished growth, vascular thickening within the kidney, and atrophy of the inner renal medulla. This phenotype is virtually identical to that seen in angiotensinogen-deficient (Agt−/−) and angiotensin-converting enzyme-deficient (Ace −/−) mice that are unable to synthesize angiotensin II. Agtr1a −/−Agtr1b −/− mice have no systemic pressor response to infusions of angiotensin II, but they respond normally to another vasoconstrictor, epinephrine. Blood pressure is reduced substantially in the Agtr1a −/− Agtr1b −/− mice and following administration of an angiotensin converting enzyme inhibitor, their blood pressure increases paradoxically. We suggest that this is a result of interruption of AT2-receptor signaling. In summary, our studies suggest that both AT1 receptors promote somatic growth and maintenance of normal kidney structure. The absence of either of the AT1 receptor isoforms alone can be compensated in varying degrees by the other isoform. These studies reaffirm and extend the importance of AT1 receptors to mediate physiological functions of the renin–angiotensin system.

Systemic hypoxia changes the organ-specific distribution of vascular endothelial growth factor and its receptors

Vascular endothelial growth factor (VEGF) plays a key role in physiological blood vessel formation and pathological angiogenesis such as tumor growth and ischemic diseases. Hypoxia is a potent inducer of VEGF in vitro. Here we demonstrate that VEGF is induced in vivo by exposing mice to systemic hypoxia. VEGF induction was highest in brain, but also occurred in kidney, testis, lung, heart, and liver. In situ hybridization analysis revealed that a distinct subset of cells within a given organ, such as glial cells and neurons in brain, tubular cells in kidney, and Sertoli cells in testis, responded to the hypoxic stimulus with an increase in VEGF expression. Surprisingly, however, other cells at sites of constitutive VEGF expression in normal adult tissues, such as epithelial cells in the choroid plexus and kidney glomeruli, decreased VEGF expression in response to the hypoxic stimulus. Furthermore, in addition to VEGF itself, expression of VEGF receptor-1 (VEGFR-1), but not VEGFR-2, was induced by hypoxia in endothelial cells of lung, heart, brain, kidney, and liver. VEGF itself was never found to be up-regulated in endothelial cells under hypoxic conditions, consistent with its paracrine action during normoxia. Our results show that the response to hypoxia in vivo is differentially regulated at the level of specific cell types or layers in certain organs. In these tissues, up- or down-regulation of VEGF and VEGFR-1 during hypoxia may influence their oxygenation after angiogenesis or modulate vascular permeability.

Hypersensitivity of Ku80-deficient cell lines and mice to DNA damage: The effects of ionizing radiation on growth, survival, and development

We recently have shown that mice deficient for the 86-kDa component (Ku80) of the DNA-dependent protein kinase exhibit growth retardation and a profound deficiency in V(D)J (variable, diversity, and joining) recombination. These defects may be related to abnormalities in DNA metabolism that arise from the inability of Ku80 mutant cells to process DNA double-strand breaks. To further characterize the role of Ku80 in DNA double-strand break repair, we have generated embryonic stem cells and pre-B cells and examined their response to ionizing radiation. Ku80−/− embryonic stem cells are more sensitive than controls to γ-irradiation, and pre-B cells derived from Ku80 mutant mice display enhanced spontaneous and γ-ray-induced apoptosis. We then determined the effects of ionizing radiation on the survival, growth, and lymphocyte development in Ku80-deficient mice. Ku80−/− mice display a hypersensitivity to γ-irradiation, characterized by loss of hair pigmentation, severe injury to the gastrointestinal tract, and enhanced mortality. Exposure of newborn Ku80−/− mice to sublethal doses of ionizing radiation enhances their growth retardation and results in the induction of T cell-specific differentiation. However, unlike severe combined immunodeficient mice, radiation-induced T cell development in Ku80−/− mice is not accompanied by extensive thymocyte proliferation. The response of Ku80-deficient cell lines and mice to DNA-damaging agents provides important insights into the role of Ku80 in growth regulation, lymphocyte development, and DNA repair.

Mannose-6-phosphate/insulin-like growth factor-II receptor is a receptor for retinoic acid

Retinoic acid (RA) exerts diverse biological effects in the control of cell growth in embryogenesis and oncogenesis. These effects of RA are thought to be mediated by the nuclear retinoid receptors. Mannose-6-phosphate (M6P)/insulin-like growth factor-II (IGF-II) receptor is a multifunctional membrane glycoprotein that is known to bind both M6P and IGF-II and function primarily in the binding and trafficking of lysosomal enzymes, the activation of transforming growth factor-β, and the degradation of IGF-II. M6P/IGF-II receptor has recently been implicated in fetal development and carcinogenesis. Despite the functional similarities between RA and the M6P/IGF-II receptor, no direct biochemical link has been established. Here, we show that the M6P/IGF-II receptor also binds RA with high affinity at a site that is distinct from those for M6P and IGF-II, as identified by a photoaffinity labeling technique. We also show that the binding of RA to the M6P/IGF-II receptor enhances the primary functions of this receptor. The biological consequence of the interaction appears to be the suppression of cell proliferation and/or induction of apoptosis. These findings suggest that the M6P/IGF-II receptor mediates a RA response pathway that is important in cell growth regulation. This discovery of the interaction of RA with the M6P/IGF-II receptor may have important implications for our understanding of the roles of RA and the M6P/IGF-II receptor in development, carcinogenesis, and lysosomal enzyme-related diseases.

Hyperactivity and impaired response habituation in hyperdopaminergic mice

Abnormal dopaminergic transmission is implicated in schizophrenia, attention deficit hyperactivity disorder, and drug addiction. In an attempt to model aspects of these disorders, we have generated hyperdopaminergic mutant mice by reducing expression of the dopamine transporter (DAT) to 10% of wild-type levels (DAT knockdown). Fast-scan cyclic voltammetry and in vivo microdialysis revealed that released dopamine was cleared at a slow rate in knockdown mice, which resulted in a higher extracellular dopamine concentration. Unlike the DAT knockout mice, the DAT knockdown mice do not display a growth retardation phenotype. They have normal home cage activity but display hyperactivity and impaired response habituation in novel environments. In addition, we show that both the indirect dopamine receptor agonist amphetamine and the direct agonists apomorphine and quinpirole inhibit locomotor activity in the DAT knockdown mice, leading to the hypothesis that a shift in the balance between dopamine auto and heteroreceptor function may contribute to the therapeutic effect of psychostimulants in attention deficit hyperactivity disorder.

The transcriptional program of a human B cell line in response to Myc

The proto-oncogene c-myc (myc) encodes a transcription factor (Myc) that promotes growth, proliferation and apoptosis. Myc has been suggested to induce these effects by induction/repression of downstream genes. Here we report the identification of potential Myc target genes in a human B cell line that grows and proliferates depending on conditional myc expression. Oligonucleotide microarrays were applied to identify downstream genes of Myc at the level of cytoplasmic mRNA. In addition, we identified potential Myc target genes in nuclear run-on experiments by changes in their transcription rate. The identified genes belong to gene classes whose products are involved in amino acid/protein synthesis, lipid metabolism, protein turnover/folding, nucleotide/DNA synthesis, transport, nucleolus function/RNA binding, transcription and splicing, oxidative stress and signal transduction. The identified targets support our current view that myc acts as a master gene for growth control and increases transcription of a large variety of genes.

Pituitary Ets-1 and GABP bind to the growth factor regulatory sites of the rat prolactin promoter

Ets factors play a critical role in oncogenic Ras- and growth factor-mediated regulation of the proximal rat prolactin (rPRL) promoter in pituitary cells. The rPRL promoter contains two key functional Ets binding sites (EBS): a composite EBS/Pit-1 element located at –212 and an EBS that co-localizes with the basal transcription element (BTE, or A-site) located at –96. Oncogenic Ras exclusively signals to the –212 site, which we have named the Ras response element (RRE); whereas the response of multiple growth factors (FGFs, EGF, IGF, insulin and TRH) maps to both EBSs. Although Ets-1 and GA binding protein (GABP) have been implicated in the Ras and insulin responses, respectively, the precise identity of the pituitary Ets factors that specifically bind to the RRE and BTE sites remains unknown. In order to identify the Ets factor(s) present in GH4 and GH3 nuclear extracts (GH4NE and GH3NE) that bind to the EBSs contained in the RRE and BTE, we used EBS-RRE and BTE oligonucleotides in electrophoretic mobility shift assays (EMSAs), antibody supershift assays, western blot analysis of partially purified fractions and UV-crosslinking studies. EMSAs, using either the BTE or EBS-RRE probes, identified a specific protein–DNA complex, designated complex A, which contains an Ets factor as determined by oligonucleotide competition studies. Using western blot analysis of GH3 nuclear proteins that bind to heparin–Sepharose, we have shown that Ets-1 and GABP, which are MAP kinase substrates, co-purify with complex A, and supershift analysis with specific antisera revealed that complex A contains Ets-1, GABPα and GABPβ1. In addition, we show that recombinant full-length Ets-1 binds equivalently to BTE and EBS-RRE probes, while recombinant GABPα/β preferentially binds to the BTE probe. Furthermore, comparing the DNA binding of GH4NE containing both Ets-1 and GABP and HeLa nuclear extracts devoid of Ets-1 but containing GABP, we were able to show that the EBS-RRE preferentially binds Ets-1, while the BTE binds both GABP and Ets-1. Finally, UV-crosslinking experiments with radiolabeled EBS-RRE and BTE oligonucleotides showed that these probes specifically bind to a protein of ∼64 kDa, which is consistent with binding to Ets-1 (54 kDa) and/or the DNA binding subunit of GABP, GABPα (57 kDa). These studies show that endogenous, pituitary-derived GABP and Ets-1 bind to the BTE, whereas Ets-1 preferentially binds to the EBS-RRE. Taken together, these data provide important insights into the mechanisms by which the combination of distinct Ets members and EBSs transduce differential growth factor responses.

Plant growth in elevated CO2 alters mitochondrial number and chloroplast fine structure

With increasing interest in the effects of elevated atmospheric CO2 on plant growth and the global carbon balance, there is a need for greater understanding of how plants respond to variations in atmospheric partial pressure of CO2. Our research shows that elevated CO2 produces significant fine structural changes in major cellular organelles that appear to be an important component of the metabolic responses of plants to this global change. Nine species (representing seven plant families) in several experimental facilities with different CO2-dosing technologies were examined. Growth in elevated CO2 increased numbers of mitochondria per unit cell area by 1.3–2.4 times the number in control plants grown in lower CO2 and produced a statistically significant increase in the amount of chloroplast stroma (nonappressed) thylakoid membranes compared with those in lower CO2 treatments. There was no observable change in size of the mitochondria. However, in contrast to the CO2 effect on mitochondrial number, elevated CO2 promoted a decrease in the rate of mass-based dark respiration. These changes may reflect a major shift in plant metabolism and energy balance that may help to explain enhanced plant productivity in response to elevated atmospheric CO2 concentrations.

Transforming Growth Factor-β1 Mediates Epithelial to Mesenchymal Transdifferentiation through a RhoA-dependent Mechanism

Transforming growth factor-β1 (TGF-β) can be tumor suppressive, but it can also enhance tumor progression by stimulating the complex process of epithelial-to-mesenchymal transdifferentiaion (EMT). The signaling pathway(s) that regulate EMT in response to TGF-β are not well understood. We demonstrate the acquisition of a fibroblastoid morphology, increased N-cadherin expression, loss of junctional E-cadherin localization, and increased cellular motility as markers for TGF-β–induced EMT. The expression of a dominant-negative Smad3 or the expression of Smad7 to levels that block growth inhibition and transcriptional responses to TGF-β do not inhibit mesenchymal differentiation of mammary epithelial cells. In contrast, we show that TGF-β rapidly activates RhoA in epithelial cells, and that blocking RhoA or its downstream target p160ROCK, by the expression of dominant-negative mutants, inhibited TGF-β–mediated EMT. The data suggest that TGF-β rapidly activates RhoA-dependent signaling pathways to induce stress fiber formation and mesenchymal characteristics.

Multifaceted Physiological Response Allows Yeast to Adapt to the Loss of the Signal Recognition Particle-dependent Protein-targeting PathwayD⃞

Translational control has recently been recognized as an important facet of adaptive responses to various stress conditions. We describe the adaptation response of the yeast Saccharomyces cerevisiae to the loss of one of two mechanisms to target proteins to the secretory pathway. Using inducible mutants that block the signal recognition particle (SRP) pathway, we find that cells demonstrate a physiological response to the loss of the SRP pathway that includes specific changes in global gene expression. Upon inducing the loss of the SRP pathway, SRP-dependent protein translocation is initially blocked, and cell growth is considerably slowed. Concomitantly, gene expression changes include the induction of heat shock genes and the repression of protein synthesis genes. Remarkably, within hours, the efficiency of protein sorting improves while cell growth remains slow in agreement with the persistent repression of protein synthesis genes. Our results suggest that heat shock gene induction serves to protect cells from mislocalized precursor proteins in the cytosol, whereas reduced protein synthesis helps to regain efficiency in protein sorting by reducing the load on the protein translocation apparatus. Thus, we suggest that cells trade speed in cell growth for fidelity in protein sorting to adjust to life without SRP.

A ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)-like protein from Chlorobium tepidum that is involved with sulfur metabolism and the response to oxidative stress

A gene encoding a product with substantial similarity to ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) was identified in the preliminary genome sequence of the green sulfur bacterium Chlorobium tepidum. A highly similar gene was subsequently isolated and sequenced from Chlorobium limicola f.sp. thiosulfatophilum strain Tassajara. Analysis of these amino acid sequences indicated that they lacked several conserved RubisCO active site residues. The Chlorobium RubisCO-like proteins are most closely related to deduced sequences in Bacillus subtilis and Archaeoglobus fulgidus, which also lack some typical RubisCO active site residues. When the C. tepidum gene encoding the RubisCO-like protein was disrupted, the resulting mutant strain displayed a pleiotropic phenotype with defects in photopigment content, photoautotrophic growth and carbon fixation rates, and sulfur metabolism. Most important, the mutant strain showed substantially enhanced accumulation of two oxidative stress proteins. These results indicated that the C. tepidum RubisCO-like protein might be involved in oxidative stress responses and/or sulfur metabolism. This protein might be an evolutional link to bona fide RubisCO and could serve as an important tool to analyze how the RubisCO active site developed.

Expression of the telomerase catalytic subunit, hTERT, induces resistance to transforming growth factor β growth inhibition in p16INK4A(−) human mammary epithelial cells

Failures to arrest growth in response to senescence or transforming growth factor β (TGF-β) are key derangements associated with carcinoma progression. We report that activation of telomerase activity may overcome both inhibitory pathways. Ectopic expression of the human telomerase catalytic subunit, hTERT, in cultured human mammary epithelial cells (HMEC) lacking both telomerase activity and p16INK4A resulted in gaining the ability to maintain indefinite growth in the absence and presence of TGF-β. The ability to maintain growth in TGF-β was independent of telomere length and required catalytically active telomerase capable of telomere maintenance in vivo. The capacity of ectopic hTERT to induce TGF-β resistance may explain our previously described gain of TGF-β resistance after reactivation of endogenous telomerase activity in rare carcinogen-treated HMEC. In those HMEC that overcame senescence, both telomerase activity and TGF-β resistance were acquired gradually during a process we have termed conversion. This effect of hTERT may model a key change occurring during in vivo human breast carcinogenesis.

Extragenic Suppressors of the Arabidopsis gai Mutation Alter the Dose-Response Relationship of Diverse Gibberellin Responses1

Active gibberellins (GAs) are endogenous factors that regulate plant growth and development in a dose-dependent fashion. Mutant plants that are GA deficient, or exhibit reduced GA responses, display a characteristic dwarf phenotype. Extragenic suppressor analysis has resulted in the isolation of Arabidopsis mutations, which partially suppress the dwarf phenotype conferred by GA deficiency and reduced GA-response mutations. Here we describe detailed studies of the effects of two of these suppressors, spy-7 and gar2–1, on several different GA-responsive growth processes (seed germination, vegetative growth, stem elongation, chlorophyll accumulation, and flowering) and on the in planta amounts of active and inactive GA species. The results of these experiments show that spy-7 and gar2–1 affect the GA dose-response relationship for a wide range of GA responses and suggest that all GA-regulated processes are controlled through a negatively acting GA-signaling pathway.